The dynamic structure of DNA in eukaryotes is integral to DNA transactions and therefore fundamentally linked to many types of cancer. Chromatin modifying enzymes, which catalyze an array of covalent posttranslational histone modifications, are responsible for mediating many of the structural transitions between different levels of chromatin packaging. Histone acetylation is the most extensively studied histone modification, and the only histone acetyltransferase (Esa1) required for cell viability in the yeast Saccharomyces cerevisiae resides within the nucleosome acetyltransferase of histone H4 (NuA4) complex. NuA4 is a large (1.3 MDa) multisubunit complex whose biochemistry has been the subject of extensive research, but little is know about its structure. This proposal seeks to utilize single-particle electron microscopy (EM) to address the following specific aims concerning NuA4 and its catalytic subcomplex, Piccolo: (1) develop an intermediate resolution (-10 A) cryo-EM model of the yeast NuA4 complex and study the interaction between NuA4 and its substrate, the nucleosome, (2) investigate the subunit organization of the NuA4 HAT complex and explore how this organization facilitates NuA4 function, and (3) determine the three-dimensional structure of the catalytic Piccolo subcomplex alone and bound to the nucleosome. To briefly describe the research design and methods for addressing these aims, EM will be used to characterize the NuA4, Piccolo, and Piccolo-NCP complexes to determine their three dimensional structures. Also, in a series of separate experiments for localizing NuA4 subunits, EM will characterize NuA4 subunit deletion mutants and NuA4 bound by antibodies specific for an engineered affinity tags present on targeted subunits. Additionally, EM will characterize the interaction between NuA4 and the nucleosome. Combined, these efforts will thoroughly characterize NuA4's structure and provide a framework for interpreting previous, ongoing, and future biochemical studies of NuA4 and related complexes. PUBLIC HEALTH RELEVANCE: This study will investigate how chromatin structure is regulated. No process is more vital to the cell than the regulation of chromatin structure, and like many essential cellular processes, its disruption can lead to cancer. Studies (like this one) of fundamentally important cellular processes related to cancer contribute greatly to the body of fundamental knowledge required to design effective cancer therapies.